Bacillus thuringiensis gene encoding a coleopteran-active toxin

ABSTRACT

A novel B.t. toxin gene encoding a protein toxic to coleopteran insects has been cloned from a novel coleopteran-active B. thuringiensis microbe. The DNA encoding the B.t. toxin can be used to transform various prokaryotic and eukaryotic microbes to express the B.t. toxin. These recombinant microbes can be used to control coleopteran insects in various environments.

BACKGROUND OF THE INVENTION

The most widely used microbial pesticides are derived from the bacteriumBacillus thuringiensis. This bacterial agent is used to control a widerange of leaf-eating caterpillars, Japanese beetles and mosquitos.Bacillus thuringiensis produces a proteinaceous paraspore or crystalwhich is toxic upon ingestion by a susceptible insect host. For example,B. thuringiensis var. kurstaki HD-1 produces a crystal called a deltatoxin which is toxic to the larvae of a number of lepidopteran insects.The cloning and expression of this B.t. crystal protein gene inEscherichia coli has been described in the published literature(Schnepf, H. E. and Whitely, H. R. [1981] Proc. Natl. Acad. Sci. U.S.A.78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 bothdisclose the expression of B.t. crystal protein in E. coli. EuropeanPatent Application, Publication No. 0 202 739, discloses a novel B.thuringiensis microbe which can be used to control coleopteran pests invarious environments.

BRIEF SUMMARY OF THE INVENTION

Disclosed and claimed is a novel toxin gene toxic to coleopteraninsects. This toxin gene can be transferred to suitable hosts viaplasmid vector.

Specifically, the invention comprises a novel delta endotoxin gene whichencodes a 74.228 kd protein which is active against coleopteran pests.

More specifically, the subject invention concerns a novel toxin gene(DNA) encoding a novel protein having activity against coleopteraninsects. Table 1 discloses the DNA encoding the novel toxin. Table 2discloses the amino acid sequence of the novel hybrid toxin. Table 3 isa composite of Tables 1 and 2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide sequence of novel toxin-encoding gene. The ORF startsas marked with arrow.

FIG. 2. Deduced amino acid sequence of novel toxin.

FIG. 3. Nucleotide sequence and corresponding amino acid sequence ofnovel toxin.

DETAILED DISCLOSURE OF THE INVENTION

The novel toxin gene of the subject invention was obtained from a novelcoleopteran-active B. thuringiensis (B.t.) isolate designated 43F. Thegene was isolated using the open reading frame (ORF) of the deltaendotoxin gene from B.t. var. san diego (B.t.s.d.) as a probe. B.t.s.d.is available from the culture repository in Peoria, Ill. U.S.A.,identified in detail, infra. where its accession number is NRRL B-15939.The gene was cloned on a 7.5 Kb EcoRI fragment in Lambda ZAP™(Stratagene Cloning Systems). This cloning vehicle readily yielded thecloned gene in the plasmid BLUESCRIPT™ (Stratagene). Sequence andexpression data are in agreement with an open reading frame of 1963 bpthat encodes a protein of 74.228 Kd.

B. thuringiensis isolate 43F, NRRL B-18298, can be cultured usingstandard known media and fermentation techniques. Upon completion of thefermentation cycle, the bacteria can be harvested by first separatingthe B.t. spores and crystals from the fermentation broth by means wellknown in the art. The recovered B.t. spores and crystals can beformulated into a wettable powder, a liquid concentrate, granules orother formulations by the addition of surfactants, dispersants, inertcarriers and other components to facilitate handling and application forparticular target pests. The formulation and application procedures areall well known in the art and are used with commercial strains of B.thuringiensis (HD-1) active against Lepidoptera, e.g., caterpillars.B.t. isolate 43F can be used to control coleopteran pests.

Subcultures of B.t. isolate 43F and the E. coli host harboring the toxingene of the invention, E. coli XL1-blue (pM1,98-4) were deposited in thepermanent collection of the Northern Research Laboratory, U.S.Department of Agriculture, Peoria, Ill., U.S.A. on Feb. 2, 1988, and onJan.15, 1988, respectively. The accession numbers are as follows:

B.t. isolate 43F--NRRL B-18298

E. coli XL1-Blue (pM1,98-4)--NRRL B-18291

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122.The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposit(s). All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

The toxin gene of the subject invention can be introduced into a widevariety of microbial hosts. Expression of the toxin gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. With suitable hosts, e.g., Pseudomonas, the microbescan be applied to the situs of coleopteran insects where they willproliferate and be ingested by the insects. The result is a control ofthe unwanted insects. Alternatively, the microbe hosting the toxin genecan be treated under conditions that prolong the activity of the toxinproduced in the cell. The treated cell then can be applied to theenvironment of target pest(s). The resulting product retains thetoxicity of the B.t. toxin.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the "phytosphere"(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type microorganisms, providefor stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Methylophilius, Agrobacterium, Acetobacter, Lactobacillus. Arthrobacter,Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast,e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Rhodotorula,and Aureobasidium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae. Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens,Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti,Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms.

A wide variety of ways are available for introducing the B.t. geneexpressing the toxin into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. One can providefor DNA constructs which include the transcriptional and translationalregulatory signals for expression of the toxin gene, the toxin geneunder their regulatory control and a DNA sequence homologous with asequence in the host organism, whereby integration will occur, and/or areplication system which is functional in the host, whereby integrationor stable maintenance will occur.

The transcriptional initiation signals will include a promoter and atranscriptional initiation start site. In some instances, it may bedesirable to provide for regulative expression of the toxin, whereexpression of the toxin will only occur after release into theenvironment. This can be achieved with operators or a region binding toan activator or enhancers, which are capable of induction upon a changein the physical or chemical environment of the microorganisms. Forexample, a temperature sensitive regulatory region may be employed,where the organisms may be grown up in the laboratory without expressionof a toxin, but upon release into the environment, expression wouldbegin. Other techniques may employ a specific nutrient medium in thelaboratory, which inhibits the expression of the toxin, where thenutrient medium in the environment would allow for expression of thetoxin. For translational initiation, a ribosomal binding site and aninitiation codon will be present.

Various manipulations may be employed for enhancing the expression ofthe messenger, particularly by using an active promoter, as well as byemploying sequences, which enhance the stability of the messenger RNA.The initiation and translational termination region will involve stopcodon(s), a terminator region, and optionally, a polyadenylation signal.

In the direction of transcription, namely in the 5' to 3' direction ofthe coding or sense sequence, the construct will involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5' or 3' of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop codon(s), thepolyadenylation signal sequence, if any, and the terminator region. Thissequence as a double strand may be used by itself for transformation ofa microorganism host, but will usually be included with a DNA sequenceinvolving a marker, where the second DNA sequence may be joined to thetoxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selectionof those hosts which have been modified or transformed. The marker willnormally provide for selective advantage, for example, providing forbiocide resistance, e.g., resistance to antibiotics or heavy metals;complementation, so as to provide prototropy to an auxotrophic host, orthe like. Preferably, complementation is employed, so that the modifiedhost may not only be selected, but may also be competitive in the field.One or more markers may be employed in the development of theconstructs, as well as for modifying the host. The organisms may befurther modified by providing for a competitive advantage against otherwild-type microorganisms in the field. For example, genes expressingmetal chelating agents, e.g., siderophores, may be introduced into thehost along with the structural gene expressing the toxin. In thismanner, the enhanced expression of a siderophore may provide for acompetitive advantage for the toxin-producing host, so that it mayeffectively compete with the wild-type microorganisms and stably occupya niche in the environment.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 basepairs (bp), preferably atleast about 100 bp, and usually not more than about 1000 bp of asequence homologous with a sequence in the host. In this way, theprobability of legitimate recombination is enhanced, so that the genewill be integrated into the host and stably maintained by the host.Desirably, the toxin gene will be in close proximity to the geneproviding for complementation as well as the gene providing for thecompetitive advantage. Therefore, in the event that a toxin gene islost, the resulting organism will be likely to also lose thecomplementing gene and/or the gene providing for the competitiveadvantage, so that it will be unable to compete in the environment withthe gene retaining the intact construct.

A large number of transcriptional regulatory regions are available froma wide variety of microorganism hosts, such as bacteria, bacteriophage,cyanobacteria, algae, fungi, and the like. Various transcriptionalregulatory regions include the regions associated with the trp gene, lacgene, gal gene, the lambda left and right promoters, the Tac promoter,the naturally-occurring promoters associated with the toxin gene, wherefunctional in the host. See for example, U.S. Pat. Nos. 4,332,898,4,342,832 and 4,356,270. The termination region may be the terminationregion normally associated with the transcriptional initiation region ora different transcriptional initiation region, so long as the tworegions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmidwill be employed which has a replication system which is functional inthe host. The replication system may be derived from the chromosome, anepisomal element normally present in the host or a different host, or areplication system from a virus which is stable in the host. A largenumber of plasmids are available, such as pBR322, pACYC184, RSF1010,pR01614, and the like. See for example, Olson et al., (1982) J.Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, andU.S. Pat. Nos. 4,356,270, 4,362,817, and 4,371,625.

The B. t. gene can be introduced between the transcriptional andtranslational initiation region and the transcriptional andtranslational termination region, so as to be under the regulatorycontrol of the initiation region. This construct will be included in aplasmid, which will include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, which have been describedpreviously. Where integration is desired, the plasmid will desirablyinclude a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms, when present. The transformants then can be tested forpesticidal activity.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility of toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Aeromonas, Vibrio. Desulfovibrio,Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas andAcetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes arefungi, such as Phycomycetes and Ascomycetes, which includes yeast, suchas Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, suchas Rhodotorula, Aureobasidium. Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t.i. gene intothe host, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; leaf affinity; lack of mammalian toxicity;attractiveness to pests for ingestion,: ease of killing and fixingwithout damage to the toxin; and the like. Other considerations includeease of formulation and handling, economics, storage stability, and thelike.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., and the like. Specific organismsinclude Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomycescerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis,and the like.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B.t.toxin gene, can be by chemical or physical means, or by a combination ofchemical and/or physical means, so long as the technique does notdeleteriously affect the properties of the toxin, nor diminish thecellular capability in protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as formaldehyde andglutaraldehyde; anti-infectives, such as zephiran chloride andcetylpyridinium chloride; alcohols, such as isopropyl and ethanol;various histologic fixatives, such as Bouin's fixative and Helly'sfixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H.Freeman and Company, 1967); or a combination of physical (heat) andchemical agents that preserve and prolong the activity of the toxinproduced in the cell when the cell is administered to the host animal.Examples of physical means are short wavelength radiation such asgamma-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like.

The cells generally will have enhanced structural stability which willenhance resistance to environmental conditions. Where the pesticide isin a proform, the method of inactivation should be selected so as not toinhibit processing of the proform to the mature form of the pesticide bythe target pest pathogen. For example, formaldehyde will crosslinkproteins and could inhibit processing of the proform of a polypeptidepesticide. The method of inactivation or killing retains at least asubstantial portion of the bio-availability or bioactivity of the toxin.

The cellular host containing the B.t. insecticidal gene may be grown inany convenient nutrient medium, where the DNA construct provides aselective advantage, providing for a selective medium so thatsubstantially all or all of the cells retain the B.t. gene. These cellsmay then be harvested in accordance with conventional ways.Alternatively, the cells can be treated prior to harvesting.

The B.t. cells may be formulated in a variety of ways. They may beemployed as wettable powders, granules or dusts, by mixing with variousinert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

The pesticidal concentration will vary widely depending upon the natureof the particular formulation, particularly whether it is a concentrateor to be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The

formulations will generally have from about 10² to about 10⁴ cells/mg.These formulations will be administered at about 50 mg (liquid or dry)to 1 kg or more per hectare.

The formulations can be applied to the environment of the coleopteranpest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling,or the like.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted. Example1--Cloning of Novel Toxin Gene and Transformation into Bacillusmegaterium

Total cellular DNA was prepared by growing the cells of B.t. isolate 43Fand B.t.s.d. to a low optical density (OD₆₀₀ =1.0) and recovering thecells by centrifugation. The cells were protoplasted in a buffercontaining 20% sucrose and 50 mg/ml lysozyme. The protoplasts were lysedby addition of SDS to a final concentration of 4%. The cellular materialwas precipitated overnight at 4° C. in 100 mM neutral potassiumchloride. The supernate was phenol/chloroform extracted twice and theDNA precipitated in 68% ethanol. The DNA was purified on a cesiumchloride gradient. DNA's from strains 43F and B.t.s.d. (as a standard ofreference) were digested with EcoRI and run out on a 0.8% agarose gel.The gel was Southern blotted and probed with the nick translated ORFXmnI to PstI fragment of the toxin encoding gene isolated from B.t.s.d.(this will be subsequently referred to as probe). The results showed 43Fto hybridize to probe at 7.5 Kb which is different than the standard.

Preparative amounts of 43F DNA were digested with EcoRI and run out on a0.8% agarose gel. The 7.5 Kb region of the preparative gel was isolatedand the DNA electroeluted and concentrated using an ELUTIP™-d(Schleicher and Schuell, Keene, NH) ion exchange column. A sample wasblotted and probed to verify the fragment was indeed isolated. The 7.5Kb EcoRI fragment was ligated to Lambda ZAP™ EcoRI arms. The packagedrecombinant phage were plated out with E. coli strain BB4 (StratageneCloning Systems, La Jolla, Calif.) to give high plaque density.

The plaques were screened by standard procedures with probe. The plaquesthat hybridized were purified and rescreened at a lower plaque density.The resulting phage were grown with M13 helper phage (Stratagene) andthe recombinant BLUESCRIPT™ plasmid was automatically excised andpackaged. The "phagemid" was re-infected in XL1-blue E. coli cells(Stratagene) as part of the automatic excision process. The infectedXL1-blue cells were screened for ampicillin resistance and the resultingcolonies were miniprepped to find the desired plasmid pM1,98-4. Therecombinant E. coli XL1-Blue (pM1,98-4) strain is called MR381.

The plasmid pM1,98-4 contained a 7.5 Kb EcoRI insert. To verify thatthis insert was the one of interest, a Southern blot was performed andprobed. The 7.5 Kb band hybridized with the probe, confirming that thefragment had been cloned. Restriction endonuclease analysis of the 7.5Kb EcoRI fragment with the enzymes HindIII, PstI, SpeI, BamHI and XbaIwas done to show that a coleopteran gene different than B.t.s.d. hadbeen cloned. The enzymes which cut inside the 7.5 Kb EcoRI fragment wereHindIII (twice) SpeI (twice) and PstI (once). The ORF of the 43F genecuts once with HindIII, twice with SpeI and does not cut with XbaI,EcoRI, or BamHI. In comparison to the coleopteranactive gene alreadycloned and sequenced, the 7.5 Kb EcoRI fragment shows no similarity inits restriction map. Sequence data shows an open reading frame of 1963bp with at best 70% homology to the toxin encoding gene of B.t.sd. Therecombinant BLUESCRIPT™ plasmid has been fused with the Bacillus plasmidpBC16-1SpeI and transformed into B. megaterium for expression by thefollowing procedure. The plasmid pM1,98-4 was completely digested withXbaI. The Bacillus vector pBC16-1, received from the Bacillus GeneticStock Center (Ohio State University), was terminally digested with EcoRIand then made blunt-ended by filling the 5' overhang using the Klenowfragment and deoxnucleotide triphosphates. SpeI linker was added and theresulting plasmid was called pBC16-1SpeI. This plasmid was terminallydigested with SpeI. The XbaI overhang of pM1,98-4 (XbaI linear) and theSpeI overhang of pBC16-1SpeI (SpeI linear) are complementary. The twowere fused together with T4 DNA Ligase and transformed into competent E.coli cells DH5 (BRL). Screening of tetracyclineresistant coloniesproduced the desired plasmid called pM2,18-1. This plasmid was thentransformed, using standard procedures, into B. megatarium. B.megatarium (pM2,18-1) was grown to sporulation producing crystalinclusions. Polyacrylamide gel analysis of a spore crystal preparationsuggests that an approximately 70 Kd molecular weight protein is beingproduced. This is in agreement with the molecular mass of 74.228 Kdpredicted from the amino acid sequence as deduced from the nucleotidesequence. The novel gene of the invention has homology to the B.t.s.d.toxin gene but is clearly distinguished from the B.t.s.d. gene by aunique nucleotide sequence.

Data from standard insect tests show that the novel toxin of theinvention is active against Leptinotarsa texana, a surrogate testspecies for the Colorado Potato Beetle (CPB). Novel B.t. isolate 43F hasbeen shown to be active against L. texana and CPB.

The above cloning procedures were conducted using standard proceduresunless otherwise noted.

The various methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. Theseprocedures are all described in Maniatis, T., Fritsch, E. F., andSambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York. Thus, it is within the skill of those inthe genetic engineering art to extract DNA from microbial cells, performrestriction enzyme digestions, electrophorese DNA fragments, tail andanneal plasmid and insert DNA, ligate DNA, transform cells, prepareplasmid DNA, electrophorese proteins, and sequence DNA.

The restriction enzymes disclosed herein can be purchased from BethesdaResearch Laboratories, Gaithersburg, Md., or New England Biolabs,Beverly, Mass. The enzymes are used according to the instructionsprovided by the supplier.

Plasmid pM1,98-4 containing the B.t. toxin gene, can be removed from thetransformed host microbe by use of standard well-known procedures. Forexample, E. coli XL1-Blue (pM1,98-4) can be subjected to cleared lysateisopycnic density gradient procedures, and the like, to recoverpM1,98-4.

Example 2--Insertion of Toxin Gene Into Plants

The novel gene coding for the novel insecticidal toxin, as disclosedherein, can be inserted into plant cells using the Ti plasmid fromAgrobacter tumefaciens. Plant cells can then be caused to regenerateinto plants (Zambryski, P., Joos, H., Gentello, C., Leemans, J., VanMontague, M. and Schell, J [1983] Cell 32:1033-1043). A particularlyuseful vector in this regard is pEND-4K (Klee, H. J., Yanofsky, M. F.and Nester, E. W. [1985] Bio/Technology 3:637-642). This plasmid canreplicate both in plant cells and in bacteria and has multiple cloningsites for passenger genes. The toxin gene, for example, can be insertedinto the BamHI site of pEND4K, propagated in E. coli, and transformedinto appropriate plant cells.

Example 3--Cloning of Novel B. thuringiensis Gene Into Baculoviruses

The novel gene of the invention can be cloned into baculoviruses such asAutographa californica nuclear polyhedrosis virus (AcNPV). Plasmids canbe constructed that contain the AcNPV genome cloned into a commercialcloning vector such as pUC8. The AcNPV genome is modified so that thecoding region of the polyhedrin gene is removed and a unique cloningsite for a passenger gene is placed directly behind the polyhedrinpromoter. Examples of such vectors are pGP-B6874, described by Pennocket al. (Pennock, G. D., Shoemaker, C. and Miller, L. K. [1984] Mol.Cell. Biol. 4:399-406), and pAC380, described by Smith et al. (Smith, G.E., Summers, M. D. and Fraser, M. J. [1983] Mol Cell. Biol.3:2156-2165). The gene coding for the novel protein toxin of theinvention can be modified with BamHI linkers at appropriate regions bothupstream and downstream from the coding region and inserted into thepassenger site of one of the AcNPV vectors.

As disclosed previously, the nucleotide sequence encoding the novel B.t.toxin gene is shown in Table 1. The deduced amino acid sequence is shownin Table 2.

It is well known in the art that the amino acid sequence of a protein isdetermined by the nucleotide sequence of the DNA. Because of theredundancy of the genetic code, i.e., more than one coding nucleotidetriplet (codon) can be used for most of the amino acids used to makeproteins, different nucleotide sequences can code for a particular aminoacid. Thus, the genetic code can be depicted as follows:

    ______________________________________                                        Phenylalanine (Phe)                                                                        TTK      Histidine (His)                                                                              CAK                                      Leucine (Leu)                                                                              XTY      Glutamine (Gln)                                                                              CAJ                                      Isoleucine (Ile)                                                                           ATM      Asparagine (Asn)                                                                             AAK                                      Metionine (Met)                                                                            ATG      Lysine (Lys)   AAJ                                      Valine (Val) GTL      Aspartic acid (Asp)                                                                          GAK                                      Serine (Ser) QRS      Glutamic acid (Glu)                                                                          GAJ                                      Proline (Pro)                                                                              CCL      Cysteine (Cys) TGK                                      Threonine (Thr)                                                                            ACL      Tryptophan (Trp)                                                                             TGG                                      Alanine (Ala)                                                                              GCL      Arginine (Arg) WGZ                                      Tyrosine (Tyr)                                                                             TAK      Glycine (Gly)  GGL                                      Termination signal                                                                         TAJ                                                              ______________________________________                                    

Key: Each 3-letter deoxynucleotide triplet corresponds to atrinucleotide of mRNA, having a 5'-end on the left and a 3'-end on theright. All DNA sequences given herein are those of the strand whosesequence correspond to the mRNA sequence, with thymine substituted foruracil. The letters stand for the purine or pyrimidine bases forming thedeoxynucleotide sequence.

A=adenine

G=guanine

C=cytosine

T=thymine

X=T or C if Y is A or G

X=C if Y is C or T

Y=A, G, C or T if X is C

Y=A or G if X is T

W=C or A if Z is A or G

W=C if Z is C or T

Z=A, G, C or T if W is C

Z=A or G if W is A

QR=TC if S is A, G, C or T; alternatively QR=AG if S is T or C

J=A or G

K=T or C

L=A, T, C or G

M=A, C or T

The above shows that the novel amino acid sequence of the B.t. toxin canbe prepared by equivalent nucleotide sequences encoding the same aminoacid sequence of the protein. Accordingly, the subject inventionincludes such equivalent nucleotide sequences. In addition it has beenshown that proteins of identified structure and function may beconstructed by changing the amino acid sequence if such changes do notalter the protein secondary structure (Kaiser, E. T. and Kezdy, F. J.[1984] Science 223:249-255). Thus, the subject invention includesmutants of the amino acid sequence depicted herein which do not alterthe protein secondary structure, or if the structure is altered, thebiological activity is retained to some degree.

We claim:
 1. Essentially pure DNA encoding a B.t. toxin having the aminoacid sequence shown in FIG.
 2. 2. Essentially pure DNA according toclaim 1, having the nucleotide sequence shown in FIG.
 1. 3. Arecombinant DNA transfer vector comprising DNA having all or part of thenucleotide sequence which codes for the amino acid sequence shown inFIG.
 2. 4. The DNA transfer vector, according to claim 3, transferred toand replicated in a prokaryotic or eukaryotic host.
 5. A bacterial hosttransformed to express a B.t. toxin having the amino acid sequence shownin FIG.
 2. 6. Bacillus megaterium, according to claim 5, transformedwith a plasmid vector containing the B.t. toxin gene encoding the B.t.toxin having the amino acid sequence shown in FIG.
 2. 7. E. coliXL1-Blue (pM1,98-4), having the identifying characteristics of NRRLB-18291, a host according to claim
 5. 8. A microorganism according toclaim 5, which is a species of Pseudomonas, Azotobacter, Erwinia,Serratia, Klebsiella, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter or Alcaligenes.
 9. A microorganism accordingto claim 8, wherein said microorganism is pigmented and phylloplaneadherent.
 10. An insecticidal composition comprising insecticidecontaining substantially intact, treated cells having prolongedpesticidal activity when applied to the environment of a target pest,wherein said insecticide is a polypeptide toxic to coleopteran insects,is intracellular, and is produced as a result of expression of atransformed microbe capable of expressing the B.t. toxin having theamino acid sequence shown in FIG.
 2. 11. The insecticidal composition,according to claim 10, wherein said treated cells are treated bychemical or physical means to prolong the insecticidal activity in theenvironment.
 12. The insecticidal composition, according to claim 11,wherein said cells are prokaryotes or lower eukaryotes.
 13. Theinsecticidal composition, according to claim 12, wherein saidprokaryotic cells are selected from the group consisting ofEnterobacteriaceae, Bacillaceae, Rhizobiaceae, Spirillaceae,Lactobacillaceae, Pseudomonadaceae, Azotobacteraceae, andNitrobacteraceae.
 14. The insecticidal composition, according to claim12, wherein said lower eukaryotic cells are selected from the groupconsisting of Phycomycetes, Ascomycetes, and Basidiomycetes.
 15. Theinsecticidal composition, according to claim 10, wherein said cell is apigmented bacterium, yeast, or fungus.
 16. Treated, substantially intactunicellular microorganism cells containing an intracellular toxin, whichtoxin is a result of expression of a Bacillus thuringiensis toxin genetoxic to coleopteran insects which codes for a polypeptide toxin havingthe amino acid sequence shown in FIG. 2, wherein said cells are treatedunder conditions which prolong the insecticidal activity when said cellis applied to the environment of a target insect.
 17. The cells,according to claim 16, wherein the cells are treated by chemical orphysical means to prolong the insecticidal activity in the environment.18. The cells according to claim 16, wherein said microorganism isPseudomonas and said toxin is a B.t. toxin having the amino acidsequence shown in FIG.
 2. 19. Pseudomonas cells according to claim 18,wherein said cells are treated with iodine.
 20. The cells, according toclaim 16, which are Pseudomonas fluorescens.
 21. Plasmid denotedpM1,98-4.
 22. B. megatarium (pM2,18-1), according to claim
 6. 23.Plasmid denoted pM2,18-1.